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Glycobiology, 2001, Vol. 11, No. 6 505-513
© 2001 Oxford University Press

Analysis of heparan sulfate oligosaccharides by nano-electrospray ionization mass spectrometry

R. Marshall Pope1,2, Christina S. Raska3, Suzanne C. Thorp4 and Jian Liu1,4

2Mass Spectrometry Facility, Department of Psychiatry, School of Medicine, The University of North Carolina, Chapel Hill, NC 27599, USA, 3Department of Chemistry, The University of North Carolina, Chapel Hill, NC 27599, USA, and 4Division of Medicinal Chemistry and Natural Products, School of Pharmacy, The University of North Carolina, Chapel Hill, NC 27599, USA

A highly sensitive method to identify and quantify heparan sulfate (HS) oligosaccharides by using nano-electrospray ionization mass spectrometry (nESI-MS) is described. The new approach allows us to detect approximately 50 nM of a chemically synthesized pentasaccharide with a structure of GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6SOMe (3-OH pentasaccharide). Typically, solutions were infused for a total of 5 min, at an average flow rate of 30 nl/min, and the remaining sample was recovered from the nanovial. The spectra shown were obtained by summing scans for 1–3 min. Hence, our data indicated that as little as 3 x 10–15 mole of the pentasaccharide was consumed to obtain a reasonable spectrum at the concentration as low as 50 nM. In addition, we found a linear relationship between the relative response of the molecular ion and the concentration of the analyzed 3-OH pentasaccharide, demonstrating that this approach can be used to determine the amount of HS oligosaccharides. To this end, a 3-O-sulfated pentasaccharide was prepared by incubating the 3-OH pentasaccharide with purified HS 3-O-sulfotransferase-1 and 3'-phosphoadenosine-5'-phospho[35S]sulfate. The resulting 3-O-sulfated pentasaccharide was purified and analyzed by nESI-MS. Based on the standard curve constructed with the 3-OH pentasaccharide, we calculated the concentration of the 3-O-sulfated pentasaccharide by the relative response. The result indicates that this value is very close to the value measured by [35S]sulfate radioactivity. In conclusion, nESI-MS provides both high sensitivity and the capacity to quantify HSs. This approach is likely to become a very important tool for structural analysis and sequencing of HS and heparin oligosaccharides.

1 To whom correspondence should be addressed


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